The overall hypothesis of this proposal is that pleckstrin and pleckstrin-2 play critical, and distinct roles in arterial hemostasis and thrombosis. Pleckstrin, which makes up approximately one percent of the total cellular protein within platelets and leukocytes, is a protein best known for containing the two prototypic Pleckstrin Homology (PH) domains. Following platelet activation, PKC rapidly phosphorylates pleckstrin, inducing it to bind membrane bound phospholipids such as phosphatidylinositol 4,5 bisphosphate (PIP2). Platelets also contain a widely expressed paralog of pleckstrin, called pleckstrin- 2. Although the activity of pleckstrin is regulated through protein phosphorylation, pleckstrin-2 is not a phosphoprotein, but is instead activated by binding a specific PI3K generated phospholipid, phosphatidylinositol 3,4 bisphosphate (PI3,4P2). To understand the true in vivo role of these two proteins, we have introduced disruptive mutations within the murine pleckstrin and pleckstrin-2 genes, and analyzed the platelet phenotype in genetically modified mice lacking either pleckstrin isoform. Platelets lacking either pleckstrin or pleckstirin-2 have impaired defects of PKC- or PI3K-mediated aggregation, actin assembly, and secretion. Platelets lacking both pleckstrin isoforms have an almost total defect in actin nucleation and exocytosis. Together, these data demonstrate the critical roles that pleckstrin and pleckstrin-2 play in several aspects of platelet biology. Based on our preliminary murine experiments, we hypothesize that pleckstrin isoforms contribute to platelet biology by facilitating the presentation of phospholipids to proteins that are required for actin assembly and for secretion. The overall goal of this proposal is to perform a comprehensive and systematic study of the function of pleckstrin and pleckstrin-2 within platelets, and to understand the differences between the two distinct isoforms. In Aim 1, we will determine the molecular link between the two pleckstrin isoforms and platelet actin dynamics. In Aim 2, we will formally identify how pleckstrin and pleckstrin-2 drive platelet secretion by using a combination of biochemistry and murine genetics. In Aim 3, we will analyze the structural mechanisms by which both pleckstrin isoforms mediate their effects PUBLIC HEALTH RELEVANCE: Heart disease and strokes are among the leading causes of morbidity and mortality in this country. Blood clots that form at sites of atherosclerosis are often the precipitating event for both of these diseases. Our work demonstrates that two platelet proteins, pleckstrin and pleckstrin-2 are important for the activation of platelets. We hope that a better understanding of the events that regulate platelet activation, including pleckstrin-mediated signaling, will lead to new therapeutic approaches to prevent vascular occlusion, as well as lead to a better understanding of platelet biology.